Interpretive Summary: Efficient recovery and accurate enumeration of Escherichia coli O157:H7 (a human pathogen) and Pseudomonas fluorescens (a biocontrol bacterium) from leafy greens is crucial for assessment of biocontrol efficacy. Other than the generation of antibiotic resistant strains (foodborne pathogens) or use of green fluorescent labeled mutants, there are limited options available for simultaneous recovery and enumeration of biocontrol and pathogenic bacteria. This research provides information on the recovery and assessment of E. coli O157:H7 and P. fluorescens from media and inoculated spinach using Restaino and Frampton (R & F) and E. coli O157:H7 Chromogenic Medium (RFCM). This study provides a methodology for evaluating the effectiveness of a biocontrol agent and establishes the utility of its use for other leafy green products. Accurate recovery and enumeration of biocontrol and pathogenic bacteria will promote optimal determination of biocontrol efficacy, a post-harvest food safety intervention strategy. This study validates the use of selective medium (RCPM) with specific applications for this biocontrol research.

Technical Abstract:
Control the post-harvest contamination of leafy greens by Escherichia coli O157:H7 is important for food safety. Efficient recovery and enumeration of E. coli O157:H7 and the biocontrol microbe Pseudomonas fluorescens from produce is crucial for assessment of biocontrol efficacy. Sensitive and effective selective media is an important tool for this study. We evaluated Restaino and Frampton (R & F) and E. coli O157:H7 Chromogenic Medium (RFCM) for the recovery of E. coli O157:H7 and Pseudomonas fluorescens from co-inoculated cultures relative to other media. The effects of storage time and temperatures on bacterial recovery from spinach inoculated with monocultures or both microbes were determined. Microbial counts of E. coli O157:H7 on RFCM were based on color (blue-black precipitate) in contrast to P. fluorescens, which had distinct white /translucent colonies that fluorescence under ultraviolet light. Bacterial populations from mixed cultures plated on different media and recovered ranged from 3.6-8.7 and 4.2-7.7 log CFU/ml for E. coli O157:H7 and P. fluorescens, respectively. Microbial counts on spinach ranged from 6.0-8.2 and 6.5-8.6 log CFU/g for the above bacteria; respectively. Bacterial recovery increased with storage time of 0 to 48 h, implying a possible increase of contact time for bacterial attachment. A greater recovery of E. coli O157:H7 and P. fluorescens was attained at 10 and 20 deg C than at 5 deg C. The RFCM was best suited for simultaneous recovery of E. coli O157:H7 and P. fluorescens based on color and size on media. Efficient recovery of E. coli O157:H7 and P. fluorescens from inoculated spinach can greatly enhance assessment of biocontrol efficacy as a post-harvest intervention strategy. This study shows that RFCM can be an effective tool for biocontrol research.